Oliveira A S, Migliolo L, Aquino R O, Ribeiro J K C, Macedo L L P, Bemquerer M P, Santos E A, Kiyota S, de Sales M P
Laboratório de Química eFunção de Proteínas, Departamento de Bioquímica, Centro de Biociências-UFRN, Natal-RN, Brazil.
Protein Pept Lett. 2009;16(12):1526-32. doi: 10.2174/092986609789839403.
Two trypsin inhibitors (called PdKI-3.1 and PdKI-3.2) were purified from the seeds of the Pithecellobium dumosum tree. Inhibitors were obtained by TCA precipitation, affinity chromatography on Trypsin-Sepharose and reversed-phase-HPLC. SDS-PAGE analysis with or without reducing agent showed that they are a single polypeptide chain, and MALDI-TOF analysis determined molecular masses of 19696.96 and 19696.36 Da, respectively. The N-terminal sequence of both inhibitors showed strong identity to the Kunitz family trypsin inhibitors. They were stable over a wide pH (2-9) and temperature (37 to 100 degrees C) range. These inhibitors reduced over 84% of trypsin activity with inhibition constant (Ki) of 4.20 x 10(-8) and 2.88 x 10(-8) M, and also moderately inhibited papain activity, a cysteine proteinase. PdKI-3.1 and PdKI-3.2 mainly inhibited digestive enzymes from Plodia interpunctella, Zabrotes subfasciatus and Ceratitis capitata guts. Results show that both inhibitors are members of the Kunitz-inhibitor family and that they affect the digestive enzyme larvae of diverse orders, indicating a potential insect antifeedant.
从猴耳环树的种子中纯化出两种胰蛋白酶抑制剂（分别称为PdKI - 3.1和PdKI - 3.2）。通过三氯乙酸沉淀、胰蛋白酶 - 琼脂糖亲和层析和反相高效液相色谱法获得抑制剂。在有或没有还原剂的情况下进行的SDS - PAGE分析表明它们是单条多肽链，基质辅助激光解吸电离飞行时间质谱分析确定其分子量分别为19696.96和19696.36道尔顿。两种抑制剂的N端序列与库尼茨家族胰蛋白酶抑制剂具有高度同源性。它们在较宽的pH范围（2 - 9）和温度范围（37至100摄氏度）内都很稳定。这些抑制剂能降低超过84%的胰蛋白酶活性，抑制常数（Ki）分别为4.20×10⁻⁸和2.88×10⁻⁸ M，并且还能适度抑制半胱氨酸蛋白酶木瓜蛋白酶的活性。PdKI - 3.1和PdKI - 3.2主要抑制印度谷螟、豆象和地中海实蝇肠道中的消化酶。结果表明这两种抑制剂都是库尼茨抑制剂家族的成员，并且它们会影响不同目昆虫幼虫的消化酶，表明其具有潜在的昆虫拒食剂作用。
